Fluorescent dyes are widely used as tracers for localization of biological structures by fluorescence microscopy, for quantification of analytes by fluorescence immunoassay, for flow cytometric analysis of cells, for measurement of physiological state of cells and other applications [Kanaoka Angew. Chem. Intl. Ed. Engl. 16:137 (1977); Hemmila, Clin. Chem. 31:359 (1985)]. Their primary advantages over other types of absorption dyes include the visibility of emission at a wavelength distinct from the excitation, the orders of magnitude greater detectability of fluorescence emission over light absorption, the generally low level of fluorescence background in most biological samples and the measurable intrinsic spectral properties of fluorescence polarization [Jolley, et al. Clin. Chem. 27:1190 (1981)], lifetime [U.S. Pat. No. 4,374,120] and excited state energy transfer [U.S. Pat. Nos. 3,996,345; 4,542,104].
For many applications that utilize fluorescent dyes as tracers, it is necessary to chemically react the dye with a biologically active ligand such as a cell, tissue, protein, antibody, enzyme, drug, hormone, nucleotide, nucleic acid, polysaccharide, lipid or other biomolecule to make a fluorescent ligand analog or to react the dye with natural or synthetic polymers. With these synthetic probes, the biomolecule frequently confers a specificity for a biochemical interaction that is under investigation and the fluorescent dye provides the method for detection and/or quantification of the interaction. Chemically reactive synthetic fluorescent dyes have long been recognized as essential for following these interactions [Soini & Hemmila, Clin. Chem. 25:353 (1979)]. It is also frequently desirable to employ more than one fluorescent conjugate simultaneously and to quantify the conjugates independently, requiring selective detection of each fluorescent probe. The dyes in common use are limited to a relatively small number of aromatic structures. It is an object of this invention to provide improved fluorescent dyes which have high water solubility. It is also an object of this invention to provide fluorescent tracers which can be used in conjunction with fluorescein and other commonly used fluorescent probes. It is further an object of this invention to provide dyes with the chemical reactivity necessary for conjugation to the functional groups commonly found in biomolecules, drugs, and natural and synthetic polymers. It is further an object of this invention to provide dyes whose fluorescence has low sensitivity to solution pH.
Coons and Kaplan [J. Exp. Med. 91:1 (1950)] first prepared a chemically reactive isocyanate of fluorescein and later Riggs, et al. [Am. J. Pathol. 34:1081 (1958)] introduced the more stable isothiocyanate analog of fluorescein. Fluorescein isothiocyanate (FITC) remains one of the most widely used tracers for fluorescent staining and immunoassay. Other reactive fluoresceins were prepared by Haugland [U.S. Pat. No. 4,213,904]. Virtually all fluorescence microscopes are equipped with excitation sources and filters optimized to excite and detect fluorescein emission. Fluorescein has broad emission in the visible portion of the spectrum beginning at approximately 480 nm, peaking at about 514 nm and decreasing to 10% of the peak intensity at approximately 580 nm.
The primary advantages that have permitted fluorescein isothiocyanate and its conjugates to remain the standard for microscopy and fluorescence immunoassay are a high absorbance, a high quantum yield and general ease of conjugation to biomolecules. Fluorescein has also become the predominant dye for use in the technique of flow cytometry [Lanier & Loken, J. Immunol. 132:151 (1984)].
There is a recognized need for suitable fluorophores for applications in multi-color microscopy [Khalfan, et al. (1986)], flow cytometry [Stryer, et al., U.S. Pat. No. 4,520,110; Titus, et al., J. Immunol. Methods 50:193 (1982)], immunoassays [Staines, et al., J. Histochem. Cytochem. 36:145 (1988)], and DNA sequencing [Smith, et al., Nature 321:674 (1986)]. Most of the dyes proposed for these applications have had longer wavelength emission than fluorescein. Since fluorescein has essentially no fluorescence below 490 nm, there is a clear opportunity to detect suitable fluorophores that have strong emission below this wavelength. The desirable dyes would have the following properties:
1. A high fluorescence quantum yield with a narrow emission peak at wavelengths sufficiently shorter than that of fluorescein so that the longest wavelength components of the dye emission have little or no spectral overlap with the fluorescein emission band. PA1 2. A high absorptivity as measured by extinction coefficient. Preferred are dyes that can be excited with the most intense emission lines of the common excitation sources such as the 365 nm line of the mercury arc lamp. Excitation below 365 nm is less desirable since it can result in cell injury or death in applications where fluorescence measurements are performed on living cells. Furthermore, autofluorescence of proteins, nucleic acids and other biomolecules present in cells (especially NADH which has peak absorbance at 340 nm and peak emission at 460 nm) is also increased with shorter wavelength excitation. Use of wavelengths longer than 350 nm also permits use of less expensive glass optics instead of quartz optics. PA1 3. High solubility of the dye and its reactive derivatives in aqueous solution to enhance the utility of the dye for modification of cells and biopolymers. PA1 4. High stability of the dye to excitation light, enhancing the utility of the dye for quantitative measurements and permitting extended illumination time and higher lamp intensities for increased sensitivity. PA1 5. For quantitative measurements, low sensitivity of the emission intensity to properties of the solution is necessary so that the measured signal is proportional only to the absolute quantity of dye present and is independent of environmental effects such as pH, viscosity and polarity. PA1 6. Suitability of the dye for preparation of reactive derivatives of several different types which exhibit reactivity toward a variety of chemically reactive sites. PA1 7. Intrinsically low biological activity or toxicity of the dye. PA1 1. Small Stokes' shifts, narrow emission bands and little spectral overlap with fluorescein. PA1 2. High extinction coefficients, quantum yields and photostabilities. PA1 3. High water solubility. PA1 4. Low sensitivity to pH. PA1 5. Reactivity with many of the functional groups found in biomolecules. PA1 6. Compatibility with common excitation sources.
A number of dyes have been proposed in the literature that can be excited and detected at wavelengths less than 500 nm. Several chemically reactive fluorophores that can be excited in the near ultraviolet and short wavelength visible region of the spectrum have been described. The spectral properties and relative water solubilities of representative examples of these dyes and compound 12 of this invention are compared in Table 1. Undesirable properties of these dyes are indicated with an asterisk. These proposed tracers are derived from naphthalene derivatives such as 5-dimethylaminonaphthalene-1-sulfonic acid (Dansyl) [Weber, Biochem. J. 41:145 (1952); Rinderknecht, Experientin 16:430 (1960)], 3-(isothiocyanato)naphthalene-1,5-disulfonic acid [Braunitzer, et al., Hoppe-Seyler's Z. Physiol. Chem. 352:1730 (1971)] and N-(4-methylphenyl)-5-isothiocyanato-1,8-naphthalimide [Khalaf & Rimpler, Hoppe-Seyler's Z. Physiol. Chem. 358:505 (1977)], pyrene derivatives such as pyrene-1-butyric acid (PBA) [Malencik & Anderson, Biochemistry 11:3022 (1972)], 8-isothiocyanatopyrene-1,3,6-trisulfonic acid (IPTS) [Braunitzer, et al., Hoppe-Seyler's Z. Physiol. Chem. 354:1536 (1973)] and 8-hydroxypyrene-1,3,6-trisulfonyl chloride [Wolfbeis, Fresenies Z. Anal. Chem. 320:271 (1985)], stilbene derivatives such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) [Cornelisse & Ploem, J. Histochem. Cytochem. 24:72 (1976)], and coumarin dyes such as 3-carboxy-7-hydroxycoumarin [Baker & Collis, J. Chem. Soc. 1949, S 12 (1949)], 7-hydroxy-4-methylumbelliferone-3-acetic acid (4-MUA) [Khalfan, et al., Biochem. J. 209:265 (1983)], 7-amino-4-methylcoumarin-3-acetic acid (AMCA) [Khalfan, et al., Histochem. J. 18:497 (1986)] and 7-diethylaminocoumarin [Staines, et al. (1988)].
The existing, reactive, blue-fluorescent fluorophores generally have much weaker absorptivity (extinction coefficients of less than 20,000 cm.sup.-1 M.sup.-1 at their absorbance maxima versus greater than 25,000 cm.sup.-1 M.sup.-1 for the compounds of this invention and 75,000 cm.sup.-1 M.sup.-1 for fluorescein at its peak near 490 nm) or lower quantum yields or lower solubility in aqueous solutions than the compounds described in this invention. Many important biological applications of fluorescence exist only in aqueous solution. Most importantly, the large Stokes' shifts and wide emission band-widths of several of these dyes result in significant residual fluorescence background from the ultraviolet excited dyes at wavelengths typically used for detection of fluorescein emission (typically 515 to 525 nm). The subject dyes have uniquely low background fluorescence at the wavelengths of the fluorescein emission maximum. With the exception of 4-MUA, the potential alternative dyes are not optimally suited for excitation with the strongest emission lines of the most commonly available sources, such as the 365 nm line of the mercury arc lamp. Fluorescence of several of the dyes that are potential alternatives to the dyes of the subject invention is frequently quenched in aqueous solution, resulting in low quantum yields. The lower quantum yield decreases the detection sensitivity or requires use of disproportionately larger quantities of the less fluorescent dye.
TABLE 1 __________________________________________________________________________ Abs Em. Emission % Emission Water Max. .epsilon. Max. Bandwidth.sup.a at 514 nm.sup.b Solu- Quantum Dye (nm) (10.sup.-3 M.sup.-1 cm.sup.-1) (nm) (nm) [cm.sup.-1 ] (% of max) bility.sup.c Yield.sup.d __________________________________________________________________________ 4-MUA 360 19.1* 454 58 [2668] 20* low* med. AMCA 354 15.2* 442 57 [2858] 12* low* med. SITS 337* 37.4 438 82 [4825]* 19* high low* Dansyl.sup.e 340* 4.5* .sup. 578*.sup.e .sup. --.sup.e .sup. --.sup.e low* low* PBA 341* 45.1 377* --.sup.f 2 low* low* IPTS 373 25.6 458 78 [3634]* 38* high low* 12 399 28.0 423 50 [2168] 3.8 high high 376 23.3 __________________________________________________________________________ *These are undesirable properties of the dyes. .sup.a Full bandwidth at half peak height .sup. b Percent of maximum emission at 514 nm .sup.c Solubility of the reactive form of the dye .sup.d In aqueous solution .sup.e The spectral properties of dansyl derivatives are very sensitive t environmental effects .sup.f Alkylpyrene derivatives show multiple emission peaks
Despite their acceptance as fluorescent tracers, coumarin derivatives have some deficiencies that preclude or make more difficult some useful applications. The reactive derivatives of aminocoumarins are quite lipophilic and insoluble in aqueous preparations. Additionally, hydroxycoumarin derivatives typically exhibit pK.sub.a values near or above 7.0 and show a pH dependent absorption spectrum which decreases the fluorescence yield of the fluorophore in the physiological pH range. The subject fluorophores have low sensitivity to the pH of the solution. This property is advantageous in the quantitative determination of fluorescence, where it is desirable to minimize the number of corrections which must be included in the calibration. Potential alternative dyes such as hydroxypyrenetrisulfonyl chloride and 4-MUA have high sensitivity to solution pH in the physiological range (pH 6 to 8).
While having other desirable properties of high absorbance and high water solubility, SITS has a very low fluorescence yield in water and is photolytically isomerized to the non-fluorescent cis isomer. Despite the commericial availability of SITS for over 15 years, use of SITS to form fluorescent conjugates has not been widely adopted due to the low fluorescence yield. SITS and related stilbene derivatives also have been found to have an inhibitory effect on anion transport systems in red blood cells [Barzilay, et al., Membrane Biochemistry 2:227 (1979)] and on microsomal glucose-6-phosphatase [Speth & Schulze, Eur. J. Biochem. 174:111 (1988)]. Another drawback of SITS is the short wavelength absorption maximum (&lt;350 nm). Ultraviolet excitation can result in cell injury and death in applications where fluorescence measurements are performed on living cells. Autofluorescence of proteins, nucleic acids and other biomolecules present in cells is also increased with shorter wavelength excitation.
Furthermore, the emission spectra of stilbene, napthalene and coumarin derivatives have a very broad long wavelength component which greatly increases the fluorescence background at wavelengths used for detection of other dyes such as fluorescein, Lucifer Yellow and tetramethylrhodamine in applications such as DNA sequencing, developmental tracers and flow cytometry that require detection of multiple dyes and dye conjugates.
With the exception of SITS and the isothiocyanate of pyrenetrisulfonic acid, the solubility in aqueous solution of the commonly used reactive forms of these dyes is very low, necessitating use of organic solvent co-mixtures in forming dye conjugates with most biopolymers. Reactive dye derivatives such as succinimidyl pyrene-1-butyrate and the succinimidyl ester of AMCA are quite lipophilic and insoluble in the aqueous solutions which are required for fluorescent labelling of proteins, polysaccharides and other biomolecules.
The high ionic charge of the pyrenyloxy sulfonic acid dyes that are the subject of this invention results in fluorescent derivatives that are highly water soluble. The ionic charge and general lack of solubility in non-polar solvents of the subject dyes enhance their usefulness as fluorescent tracers for hydrophilic environments and increase their suitability for use in applications requiring neural and developmental tracers. This property also facilitates the coupling, in aqueous solution, of the fluorescent dye and a protein, drug, or other ligand of interest. Another potential advantage is the low toxicity of closely related compounds such as 8-hydroxypyrene-1,3,6-trisulfonic acid, which has been reported to have an LD.sub.50 exceeding 1000 mg/kg [Lutty, Toxicol. Appl. Pharmacol. 44:225 (1978)].
In conclusion, the dyes which are the subject of this invention exhibit all of the desirable properties described above, namely: